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Evaluation of the Dermal Irritancy of Chemicals
Published in David W. Hobson, Dermal and Ocular Toxicology, 2020
Enzymes are protein catalysts of biological origin, a catalyst being defined as a substance which increases the rate of a particular chemical reaction without itself being consumed or permanently altered.71 Knowledge of enzymes and their actions goes back at least 150 years. Clinical enzymology, the application of the knowledge of enzymes to the diagnosis and treatment of disease processes, is a still rapidly developing field in contemporary clinical chemistry. Measurements of digestive enzyme activity in body fluid as an aid to diagnosis date back to the early 1900s. Measurement of enzyme activity in serum began in the 1920s and 1930s with studies on alkaline phosphatase in bone and liver diseases, and acid phosphatase in prostatic cancer. In 1943, Warburg and Christian observed increased activities of glycolytic enzymes in sera of tumor-bearing rats, and, in 1955, LaDue et al. reported a transitory rise of glutamic-oxaloacetic transaminase (SGOT; now called aspartate transaminase, AST) activity in serum following acute myocardial infarction.71
Introduction
Published in Clive R. Bagshaw, Biomolecular Kinetics, 2017
Kinetics is the core technique of enzymology, the study of Nature’s catalysts. It both defines the magnitude of the problem under study (i.e., the degree to which a reaction is speeded up over the noncatalyzed reaction) as well as provides a framework for its solution (i.e., the characterization of the alternative pathway(s) taken [8]). Every general biochemistry textbook devotes a section to the fundamentals of enzyme kinetics and describes how such measurements led to the concept of an enzyme–substrate Michaelis complex [9,10], long before detailed structural information became available. Identification of intermediates and measurement of their lifetimes remains the primary goal of kinetic assays. To this end, kinetic measurements are usually interpreted in conjunction with structural data to help define the mechanism in chemical terms. However, kinetic methods are used to study processes other than catalysis, with ligand binding and macromolecular folding being among other topics that are addressed here.
Plant Phenolics
Published in Ruth G. Alscher, John L. Hess, Antioxidants in Higher Plants, 2017
Thus, a body of evidence in other plant species exists in support of conversion of phenylpropanoid metabolites, such as caffeic acid 7 into protocatechuic 8 and gallic 1 acids. Additional support for an indirect pathway comes from studies using Lithospermum erythrorhizon cell cultures, where it was recently established that p-hydroxybenzoic acid 13 was derived from/j-coumaric acid 10.10 This transformation was proposed to proceed via a "nonoxidative" pathway rather than by "β-oxidation", although the enzymes were not identified. A similar situation might be envisaged for gallic 1 and protocatechuic 8 acids. Therefore, in light of such findings, there is a need to clarify the relative importance of both direct and indirect, i.e., "β-oxidation" or "nonoxidative", routes in protocatechuic 8 and gallic 1 acid biogenesis. This understanding will require precise definition of enzymology, and the subcellular location of enzymes and metabolites.
Mixed and non-competitive enzyme inhibition: underlying mechanisms and mechanistic irrelevance of the formal two-site model
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2023
Resorting to authoritative sources certainly reduces the risk of misapprehension, but for those who do not have a strong background in enzymology, attaining a proper understanding of mixed inhibition and its molecular determinants remains arduous. For example, Cornish-Bowden in “Fundamental of Enzyme Kinetics” clearly explains the limits of the formal two-site mechanism and suggests that mixed inhibition occurs mainly as a case of product inhibition with iso-mechanism enzymes12. Johnson, in “Kinetic Analysis for the New Enzymology”, adds that mixed inhibition, analogous to uncompetitive inhibition, occurs mainly with multi-substrate reactions, either as a form of product inhibition or, for ternary-complex mechanisms, when a dead-end inhibitor binds before the variable substrate.7 Copeland instead, in “Evaluation of Enzyme Inhibition in Drug Discovery”, mentions five mechanisms through which active site-directed inhibitors can cause inhibition patterns typical of mixed inhibition.3 However, these five mechanisms are treated as exceptions to a general rule. With the general rule being, again, the formal two-site mechanism.
A mechanistic modelling approach for the determination of the mechanisms of inhibition by cyclosporine on the uptake and metabolism of atorvastatin in rat hepatocytes using a high throughput uptake method
Published in Xenobiotica, 2020
Simon J. Carter, Alex S. Ferecskó, Lloyd King, Karelle Ménochet, Ted Parton, Michael J. Chappell
It is increasingly common to use compartmental mechanistic models to simultaneously describe the data obtained from hepatocytes across a range of concentrations and timepoints (Baker & Parton, 2007; Grandjean et al., 2014a; Menochet et al., 2012b; Shitara & Sugiyama, 2017). Unlike the two-step method published by Ishigami et al. (1995), mechanistic models do not assume linearity over initial timepoints (Li et al., 2013), and can be used to describe the kinetics of substrate and quantifiable metabolites as well as differences in passive rates travelling to and from the cell (Grandjean et al., 2014a; Menochet et al., 2012b), to enable better predictions of human pharmacokinetics for transporter substrates (Jones et al., 2012). The use of mechanistic models derived using the Michaelis-Menten kinetics equation (termed macro-rate constant models in this article) are based on assumptions originally designed for use in enzymology and applied to transporters: It is assumed that the association to the transporter is very rapid in comparison to the dissociation from the transporter and that the amount of substrate at the transporter is much larger than the total amount of transporters and thus the substrate is at equilibrium in a very short space of time (Grandjean et al., 2014a; Segel, 1993b). It then follows that the rate of translocation into the cell is the rate limiting step in the movement of substrate into the cell (Grandjean et al., 2014a; Segel, 1993b). However, these assumptions are not formally tested, but can be done so through the use of micro-rate constant mechanistic models.
Kinin B1 receptors as a therapeutic target for inflammation
Published in Expert Opinion on Therapeutic Targets, 2018
Fatimunnisa Qadri, Michael Bader
One particularly important family of inflammatory mediators playing an integral role during inflammation is the kinins. Kinins are blood- and tissue-derived vasoactive hormones and consist mainly of the nonapeptide, bradykinin (BK, Arg–Pro–Pro–Gly–Phe–Ser–Pro–Phe–Arg), the decapeptide Lys-bradykinin or kallidin (KD), and their carboxy-terminal des-Arg metabolites, des-Arg9-BK (DABK) and des-Arg10-KD (DAKD), respectively. There are two classical pathways for the generation of kinins, the plasma and tissue kallikrein–kinin system (KKS) (Figure 1). Kinins originate from kininogens (high and low molecular weight), which are circulatory glycoproteins primarily synthetized by the liver. The cleavage of kininogens by the proteolytic enzymes, kallikreins, in either plasma or tissue produces the kinins, BK, and KD, respectively. Both BK and KD are highly instable peptides and can be degraded very fast by several kininases including angiotensin-converting enzyme (ACE), neutral endopeptidase (NEP), carboxypeptidase N (CPN), and carboxypeptidase M (CPM). These kininases are divided into two main types on the basis of their enzymology; kininase-I (CPN and CPM), and kininase-II (ACE). Kininase-I enzymes cleave the carboxyterminal arginine from either BK or KD giving rise to the active metabolites, DABK and DAKD, while the kininases-II cleave off the C-terminal dipeptide Phe–Arg [2–6].